RNA plays a central role in many aspects of biochemistry. To carry out these cellular functions, it is critical that RNAs maintain their complex secondary, tertiary and quaternary structures. Furthermore, it is becoming evident that RNAs readily interconvert between two or more stable (or pseudo-stable) structures as part of their normal biological function. These rearrangements can be involved in control of ribozyme activity, as in the case of docking, or translational control of an mRNA through formation of alternative structures in the 5'- or 3'-UTRs. This project revolves around understanding the thermodynamic basis for the structural changes observed in certain RNAs. One type of structural change we focus on is the process of cold denaturation, where RNAs unfold at low temperature. Of particular importance to these processes, is the change in heat capacity (deltaC-p) upon folding, a property previously believed to be negligible, but now shown to be significant in certain cases. The cold denaturation of the hammerhead ribozyme is the best studied to date and the focus of the first specific aim. The thermodynamics to cold denaturation will then be compared to denaturation induced by high temperature and chaotropic agents. The generality of cold denaturation will be probed as part of specific aim 2, as well as the relationship between cold denaturation and riboregulation processes involved in the cold shock response. A small non-coding RNA from E. coil called DsrA is involved in regulating this process. We are probing the structural changes of this RNA and how these structural changes affect the complexes it makes with proteins (Hfq) and other RNAs. The 3rd major goal of this proposal is to look at the underlying nature of deltaH and deltaC-p in nucleic acid folding and understand the thermodynamic contribution of metal hydration/dehydration reactions on the overall energetics of tertiary structure formation of RNAs.